URL: http://www.addgene.org/11647
Proper Citation: RRID:Addgene_11647
Insert Name: mPGK, GDNF, rtTR-KRAB-2SM2, Tet-Off
Organism: Homo sapiens
Bacterial Resistance: Ampicillin
Defining Citation: PMID:16432520
Vector Backbone Description: Backbone Size:11516; Vector Backbone:pLVPT-rtTR-KRAB-2SM2; Vector Types:Mammalian Expression, Lentiviral; Bacterial Resistance:Ampicillin
Comments: Transgenes can be expressed from any RNA Pol II promoter as part of bicistronic unit comprising the KRAB-based repressor; tetO sequences are inserted into the vector LTR. Tet-on and Tet-off versions rely on repressors that bind in the absence or the presence of doxycycline, respectively. Addition of Pol III promoter-small hairpin RNA cassette allows for drug-controllable RNA interference (Tet-on shRNA). Cloning shRNA into pLVET, pLVCT, and pLVPT vectors: first clone shRNA into pLVTHM downstream of the tetO-H1 region. Then cut pLVTHM containing your shRNA with MscI-FspI and clone the insert containing the 3'LTR to target plasmid opened with MscI-FspI. FspI cuts into AmpR. This means for inverted clones AmpR will not be restored; after selection you will be left with clones with the shRNA cassette and functional AmpR. pLVTHM and packaging plasmid for this system are also available at Addgene http://www.addgene.org/rnaitools Please visit Trono lab website http://tronolab.epfl.ch to see frequently asked questions on cloning strategies and packaging. You may also visit LentiWeb http://www.lentiweb.com for discussion on cloning strategies and protocols.
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Source: Addgene